1. Field of the Invention
This invention relates to the fields of bioluminescent labels for microanalysis and drug discovery.
2. Background
The increased demand for simultaneously detecting more than one analyte in physiological fluids has generated a greater interest in array detection methodologies. Accurate determination of several biomolecules in a sample allows a more accurate diagnosis. In these assays, the label that gives a distinguishable signal is one of the most important parts of the assay. For an array detection to be successful, each analyte must generate a distinguishable signal from the signals generated by the other analytes. Fluorescent labels have been previously used extensively in array detection. The inherent disadvantage of fluorescent detection for biological samples is the background fluorescence that is inherent with the nature of this type of labels.
Bioluminescent labels offer several advantages over fluorescence detection. Bioluminescent labels, being rare in nature, have much smaller interference from biological and other matrices. They are not also prone to photo degradation. The phenomenon of bioluminescence, unlike fluorescence, is relatively rare in biological systems, thus, the sample does not produce any significant background signal. However, the bioluminescent labels have similar emission characteristics i.e., they emit light at roughly the same wavelength. Therefore, wild-type bioluminescent labels are not useful for array detection. New proteins that are capable of producing light at different wavelengths would be very beneficial as labels in array detection.
Aequorin is a calcium sensitive photo protein isolated from the jellyfish, Aequorea Victoria. It is widely used as a label in immunoassays and to monitor intracellular levels of free calcium. The X-ray crystal structure of aequorin reveals three EF-hand Ca2+-binding sites, a hydrophobic pocket in which the chromophore, coelenterazine, resides. In the presence of molecular oxygen, coelenterazine and apoaequorin form a stable aequorin complex. The addition of calcium causes a conformational change in the aequorin. This conformational change results in the oxidation of the non-covalently bound chromophoric unit, the coelenterazine to an excited coelenteramide. As the excited coelenteramide relaxes, light is emitted at 469 nm.
Aequorea Victoria uses the blue light emitted from aequorin as an excitation light source to stimulate the emission of green light from Aequorea Victoria's more famous and scientifically ubiquitous protein, Green Fluorescent Protein (GFP). GFP has long been used as a label in various scientific fields. Extensive research concerning GFP's structural and photochemical properties has resulted in the production of many spectrally shifted GFP mutants. The spectral shifts in GFP have proved extremely useful, allowing for single well multiple analyte analysis, multicolor reporting of cellular processes, and FRET measurements to study protein-protein interactions Bacskai et al., J Biomed Opt. 2003 Jul; 8(3): 368-75.
An advantage of using aequorin and other bioluminescent proteins instead of GFP is that since the bioluminescence emitted by these proteins is measured over virtually zero background, the proteins can be detected at extremely low levels (levels less than 10−18 moles). Additionally, this photoprotein retains its bioluminescence in a variety of buffers with a number of different additives. It can be stored in solution at 4° C. for over a month while still retaining 85% of its original activity. Lyophilization of the protein allows for its storage up to one year. Accordingly, the creation of aequorin variants with significantly different emission maximum would result in a biochemical label that is superior to GFP.
Aequorin has been used extensively, most notably for detection of calcium concentrations in vivo and as a label in immunoassays and nucleic acid probe-based assays. It has been demonstrated that aequorin functions as highly sensitive labels in the determination of both large (Erikaku, Biochem Biophys Res Commun 1991, 174,1331-6; Zenno, Biochem Biophys Res Commun 1990, 171, 169-74; Jackson, J Immunol Methods 1996, 190, 189-97; Yeh, Anal Biochem 1996, 236, 126-33) and small biomolecules (Yan, Anal Biochem 1994, 223, 111-8; Feltus, Anal Biochem 1997, 254, 62-; Ramanathan, Analytica Chemica Acta 1998, 369, 181-188). Some examples of binding assays employing aequorin as a label include the determination of amplified cytokine products (Xiao, J Immunol Methods 1996, 199, 139-47), human chorionic gonadotropin, testosterone, thyrotropin (Sgoutos, Clin. Chem. 1995, 41, 1637-1643) and human tumor necrosis factor-α (Erikaku, Biochem Biophys Res Commun 1991, 174, 1331-6). A majority of these assays have been performed in a noncompetitive assay format in which the wild type photoprotein has been covalently coupled to antigenic molecules or antibodies. See also, e.g., Tsuji et al. (1986) “Site-specific mutagenesis of the calcium-binding photoprotein aequorin,” Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al. (1985) “Cloning and Expression of the cDNA Coding for Aequorin, a Bioluminescent Calcium-Binding Protein,” Biochemical and Biophysical Research Communications 126:1259-1268; Prasher et al. (1986) Methods in Enzymology 133:288-297; Prasher, et al. (1987) “Sequence Comparisons of cDNAs Encoding for Aequorin Isotypes,” Biochemistry 26:1326-1332; Charbonneau et al. (1985) “Amino Acid Sequence of the Calcium-Dependent Photoprotein Aequorin,” Biochemistry 24:6762-6771; Shimomura et al. (1981) “Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein,” Biochem. J. 199:825-828; Inouye et al. (1989) J. Biochem. 105:473-477; Inouye et al. (1986) “Expression of Apoaequorin Complementary DNA in Escherichia coli,” Biochemistry 25:8425-8429; Inouye et al. (1985) “Cloning and sequence analysis of cDNA for the luminescent protein aequorin,” Proc. Natl. Acad. Sci. USA 82:3154-3158; Prendergast, et al. (1978) “Chemical and Physical Properties of Aequorin and the Green Fluorescent Protein Isolated from Aequorea forskalea” J. Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 064 A1; European Patent Application 0 226 979 A2, European Patent Application 0 245 093 A1 and European Patent Specification 0 245 093 B1; U.S. Pat. No. 5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQ ID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form, described in U.S. Pat. No. 5,162,227, European Patent Application 0 540 064 A1 and Sealite Sciences Technical Report No. 3 (1994).
Of particular relevance to the subject matter are U.S. Pat. Nos. 5,798,441; 5,766,941; 5,744,579; 5,541,309; 5,491,084; 5,422,266; and 5,360,728. Deo et al., Anal Biochem. 2000 May 15; 281(1):87-94; Malikova et al., FEBS Lett. 2003 Nov 6; 554(1-2):184-8; Vysotski et al., Biochemistry. 2003 May 27; 42(20):6013-24; Bondar et al., Biochemistry (Mosc). 2001 Sep; 66(9):1014-8; Kurose et al., Proc. Natl. Acad. Sci. USA, January 1999; Ohmiya et al., FEBS, vol. 301, no. 2, pp. 197-201, April 1992; Ohmiya et al., FEBS, vol. 320, no. 3, pp. 267-270, April 1993; Lewis et al., Bioconjugate Chem. 2000, 11:65-70; U.S. Pat. No. 5,876,995; all of which are incorporated by reference.
The use of aequorin as a label for multiple analyte single well analysis and multicolor reporting of cellular processes has been limited because of recombinant aequorin's single emission maximum wavelength of 469 nm. Wild type aequorin emits at a constant wavelength regardless which coelenterazine variant is used. By using different chromophore analogues, incorporation of non-natural amino acids and site-directed mutagenesis, the inventors have discovered a number of mutants that can be used to make superior biochemical labels because they have an bioluminescent emission shifted with respect to wild type aequorin.
Obelin is another bioluminescent protein from the marine hydroid Obelia longissima consisting of a single 22.2 kDA polypeptide chain. The primary sequences of obelin, aequorin and other photoproteins are highly homologous so presumably generate bioluminescence by a common chemical mechanism. There is also sequence homology in regions corresponding to the EF-hand structures of calcium-binding proteins such as calmodulin and troponin C, suggesting that some resemblance in three dimensional structures might also ensue. Obelin, along with a number of other photoproteins, is available in an efficient expression system.
All references cited within this document are explicitly incorporated by reference for all purposes.